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Abstract:

In one embodiment, the invention is to a sample metering device,
comprising a sample holding chamber oriented between a sample entry port
and a sample extraction unit, wherein a portion of said extraction unit
defines a metered volume of a sample. A diluent may be transported over
and/or through the extraction unit to form a diluted sample for sample
analysis. In another embodiment, the invention is to an apparatus and
method for rapid determination of analytes in liquid samples by various
assays including immunoassays incorporating a sample dilution feature,
capable of being used in the point-of-care diagnostic field is provided.
The devices and methods of the invention preferably are well-suited for
high range sample dilution.

Claims:

1. A sample metering device, comprising: a sample holding chamber
oriented between a sample entry port and a sample extraction unit,
wherein a portion of said sample extraction unit defines a metered volume
of a sample.

2. The device of claim 1, wherein a distal portion of said extraction
unit defines said metered volume of said sample.

3. The device of claim 1, wherein the sample is selected from the group
consisting of blood, plasma, serum, urine, interstitial fluid and
cerebrospinal fluid.

15. A method of performing an assay for an analyte in a sample, said
method comprising the steps of: introducing a sample into a sample
chamber of a cartridge, wherein the sample chamber terminates in a sample
extraction unit; loading said extraction unit with said sample; washing a
portion of said sample from the extraction unit using a volume of diluent
from a diluent conduit to form a diluted sample; and analyzing said
diluted sample for an analyte.

16. The method of claim 15, wherein the volume of diluent is a metered
volume of diluent.

17. The method of claim 15, further comprising: transporting said diluted
sample to a sensor; and performing an analyte assay at said sensor.

18. The method of claim 15, wherein the diluted sample has a dilution
ratio of from about 50:1 to about 50,000:1 (v/v diluent:sample).

Description:

PRIORITY CLAIM

[0001] The present application is a divisional application of U.S. patent
application Ser. No. 13/308,943, filed Dec. 1, 2011 which claims priority
to U.S. Provisional Application No. 61/419,489, filed Dec. 3, 2010, the
entire contents and disclosures of which are hereby incorporated by
reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to devices and methods for
rapid determination of analytes in liquid samples by various assay
techniques including immunoassays incorporating a sample dilution
feature, which preferably is suitable for high range sample dilution. The
apparatus preferably is capable of being used in the point-of-care
diagnostic field, including, for example, use at accident sites,
emergency rooms, in surgery, in intensive care units, and also in
non-medical environments. The invention is also directed to novel sample
metering devices for use in such devices and methods.

BACKGROUND OF THE INVENTION

[0003] A multitude of laboratory immunoassay tests for analytes of
interest are performed on biological samples for diagnosis, screening,
disease staging, forensic analysis, pregnancy testing and drug testing,
among others. While a few qualitative tests, such as pregnancy tests,
have been reduced to simple kits for a patient's home use, the majority
of quantitative tests still require the expertise of trained technicians
in a laboratory setting using sophisticated instruments. Laboratory
testing increases the cost of analysis and delays the patient's receipt
of the results. In many circumstances, this delay can be detrimental to
the patient's condition or prognosis, such as for example the analysis of
markers indicating myocardial infarction and heart failure. In these and
similar critical situations, it is advantageous to perform such analyses
at the point-of-care, accurately, inexpensively and with minimal delay.

[0004] Many types of immunoassay devices and processes have been
described. For example, a disposable sensing device for measuring
analytes by means of immunoassay in blood is disclosed by Davis et al. in
U.S. Pat. No. 7,419,821, the entirety of which is incorporated herein by
reference. This device employs a reading apparatus and a cartridge that
fits into the reading apparatus for the purpose of measuring analyte
concentrations. A potential problem with such disposable devices is
variability of fluid test parameters from cartridge to cartridge due to
manufacturing tolerances or machine wear. U.S. Pat. No. 5,821,399 to
Zelin, the entirety of which is incorporated herein by reference,
discloses methods to overcome this problem using automatic flow
compensation controlled by a reading apparatus having conductimetric
sensors located within a cartridge.

[0005] Electrochemical detection, in which the binding of an analyte
directly or indirectly causes a change in the activity of an
electroactive species adjacent to an electrode, has also been applied to
immunoassays. For an early review of electrochemical immunoassays, see
Laurell et al., Methods in Enzymology, vol. 73, "Electroimmunoassay",
Academic Press, New York, 339, 340, 346-348 (1981).

[0006] In an electrochemical immunosensor, the binding of an analyte to
its cognate antibody produces a change in the activity of an
electroactive species at an electrode that is poised at a suitable
electrochemical potential to cause oxidation or reduction of the
electroactive species. There are many arrangements for meeting these
conditions. For example, electroactive species may be attached directly
to an analyte, or the antibody may be covalently attached to an enzyme
that either produces an electroactive species from an electroinactive
substrate or destroys an electroactive substrate. See M. J. Green (1987)
Philos. Trans. R. Soc. Lond. B. Biol. Sci. 316:135-142, for a review of
electrochemical immunosensors. Magnetic components have been integrated
with electrochemical immunoassays. See, for example, U.S. Pat. Nos.
4,945,045; 4,978,610; and 5,149,630, each to Forrest et al. Furthermore,
jointly-owned U.S. Pat. No. 7,419,821 to Davis et al. (referenced above)
and U.S. Pat. Nos. 7,682,833 and 7,723,099 to Miller et al. teach
electrochemical immunosensing devices and methods.

[0007] Microfabrication techniques (e.g., photolithography and plasma
deposition) are attractive for construction of multilayered sensor
structures in confined spaces. Methods for microfabrication of
electrochemical immunosensors, for example on silicon substrates, are
disclosed in U.S. Pat. No. 5,200,051 to Cozette et al., the entirety of
which is incorporated herein by reference. These include dispensing
methods, methods for attaching biological reagent, e.g., antibodies, to
surfaces including photoformed layers and microparticle latexes, and
methods for performing electrochemical assays.

[0008] In U.S. Pat. No. 4,946,795, Gibbons et al. disclose a sample
dilution cartridge that relies on hydrostatic pressure. Jointly-owned
U.S. Pat. No. 6,750,053 to Widrig et al., the entirety of which is
incorporated herein by reference, teaches sample metering based on a
holding chamber with a capillary stop feature.

[0009] Notwithstanding the above literature, there remains a need in the
art for improved immunosensing devices with a greater range of detection
of analytes, including, for example, analytes present at low levels such
as cardiac troponin I, and analytes present at high levels, such as CRP.
The need also exists for improved devices and methods for metering
samples, particularly in point-of-care analyte testing. These and other
needs are met by the present invention as will become clear to one of
skill in the art to which the invention pertains upon reading the
following disclosure.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to immunosensing devices and
methods of performing an immunoassay with immunosensors incorporating a
sample dilution feature preferably suitable for high range dilution,
e.g., dilutions that are greater than about 50:1 (v/v diluent:sample), to
provide diverse real-time or near real-time analysis of analytes. The
invention, in other embodiments, is directed to novel metering devices
for metering a biological sample for analysis.

[0011] In one embodiment, for example, the invention is to a sample
metering device, comprising a sample holding chamber oriented between a
sample entry port and a sample extraction unit, wherein a portion of said
extraction unit defines a metered volume of a sample. Preferably, a
distal portion of said extraction unit defines said metered volume of
said sample. The sample may vary widely, and may be selected, for
example, from the group consisting of blood, plasma, serum, urine,
interstitial fluid and cerebrospinal fluid.

[0012] In another embodiment, the invention is to a sensor cartridge for
sensing at least one analyte in a sample, comprising: at least one
sensor, e.g., an immunosensor, in an analysis conduit; a sample chamber
between a sample entry port and a sample extraction unit; a diluent
package containing a diluent; a diluent conduit configured for
transporting diluent from the diluent package to the sample extraction
unit; and a pump configured to transfer said diluent through said diluent
conduit, over and/or through said sample extraction unit, and into said
analysis conduit. A distal portion of the extraction unit preferably
defines a metered volume of the sample. Optionally, the diluent conduit
defines a metered volume of diluent. Preferably, the sensor is selected
from the group consisting of an immunosensor, an ion sensor, a metabolite
sensor, an enzymatic sensor, an enzyme activity sensor and a nucleotide
sensor. The cartridge preferably is configured to dilute said sample at a
dilution ratio of about 50:1 to about 50,000:1 (v/v diluent:sample).

[0013] In another embodiment, the invention is to a method of performing
an assay for an analyte in a sample, said method comprising the steps of:
introducing a sample into a sample chamber of a cartridge, such as the
cartridge described above, wherein the sample chamber terminates in a
sample extraction unit; loading said extraction unit with said sample;
washing a portion of said sample from the extraction unit using a volume
of diluent from a diluent conduit to form a diluted sample; and analyzing
said diluted sample for an analyte. The method preferably further
comprises the steps of transporting said diluted sample to a sensor,
e.g., with a pump; and performing an analyte assay at said sensor.
Optionally, the method further comprises a step of characterizing the
dilution ratio, e.g., with a dilution determinant marker that may be
present in the sample extraction unit, added to the sample before being
introduced into the cartridge or otherwise incorporated into the sample
during analysis.

[0014] In each of these embodiments, the form of the sample extraction
also may vary. Preferably, the sample extraction unit comprises a porous
hydrophilic material. Examples of materials suitable for the extraction
unit include a cellulose material, nitrocellulose, cotton fiber, paper,
glass-filled paper, or a transverse filter material. The extraction unit
preferably has a porous outer coating. In one aspect, the extraction unit
includes a lysing agent, such as sodium deoxycholate or saponin, and the
extraction unit may include a dilution determinant marker, preferably
suitable for verifying the dilution level of the sample. In one aspect,
the extraction unit comprises a reactant such as ferrocene monocarboxylic
acid.

[0015] The above summary of the present invention is not intended to
describe each illustrated embodiment or every implementation of the
present invention. The figures and the detailed description that follow
more particularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] These and other objectives, features and advantages of the present
invention are described in the following detailed description of the
specific embodiments and are illustrated in the following Figures, in
which:

[0017] FIGS. 1A and 1B are isometric top and bottom views, respectively,
of an immunosensor cartridge cover in accordance with one embodiment of
the present invention;

[0018] FIG. 2 is a top view of the layout of a tape gasket for an
immunosensor cartridge in accordance with one embodiment of the present
invention;

[0019] FIG. 3 is an isometric top view of an immunosensor cartridge base
in accordance with one embodiment of the present invention;

[0020] FIG. 4 is an exploded view of an immunosensor cartridge according
to one embodiment of the invention;

[0021] FIG. 5 is a schematic of the layout of an immunosensor cartridge
with an integrated sample isolation unit in accordance with one
embodiment of the present invention;

[0022] FIG. 6 is a flow chart of the fluid and air paths within an
immunosensor cartridge with an integrated sample isolation unit in
accordance with one embodiment of the present invention;

[0023] FIG. 7 illustrates a foldable cartridge housing in accordance with
one embodiment of the present invention;

[0024] FIG. 8 is a schematic of the layout of an immunosensor cartridge
with an integrated fixed sample extraction unit in accordance with one
embodiment of the present invention;

[0025] FIG. 9 is a flow chart of the fluid and air paths within an
immunosensor cartridge with an integrated fixed sample extraction unit in
accordance with one embodiment of the present invention;

[0026] FIG. 10A shows a side view of one embodiment of the immunosensor
cartridge of the present invention, and FIG. 10B shows enlarged details
of the hemolysis detection device therein;

[0027] FIG. 11 illustrates the principle of operation of an
electrochemical immunosensor;

[0028] FIG. 12 a side view of the construction of an electrochemical
immunosensor with antibody-labeled particles (not drawn to scale); and

[0029] FIG. 13 is a top view of the mask design for the conductimetric and
immunosensor electrodes for an immunosensor cartridge in accordance with
one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to devices, e.g., single-use
disposable assay cartridges, and to methods of using such devices to
determine the presence or concentration of analytes in a liquid sample.
The invention may be particularly adapted for conducting diverse
real-time or near real-time assays of analytes. In specific embodiments,
the invention relates to devices that are configured for the controlled
metered dilution of biological samples (e.g., blood, plasma, serum,
urine, interstitial fluid and cerebrospinal fluid) and of analytes in the
diluted samples using electrochemical immunosensors or other
ligand/ligand receptor-based biosensors.

[0031] In a first embodiment, the devices and methods are particularly
adapted for low range dilution of a biological sample, e.g., dilutions
that are less than about 50:1 (v/v diluent:sample). In this aspect, a
sample is metered in a sample dilution chamber to form a metered sample,
and the diluent (which may or may not be metered) is added to the metered
sample to form a diluted sample that may be subjected to biological
analysis, e.g., in an immunoassay on one or more electrodes.

[0032] In a second embodiment, the devices and methods are particularly
adapted for high range dilution, e.g., dilutions of about 50:1 or greater
(v/v diluent:sample), typically from 50:1 to 50,000:1. In this aspect, a
portion of the sample is isolated in a fixed sample extraction unit,
which preferably is formed of a wicking material. The diluent is
subsequently passed over and/or through all or a portion of the fixed
sample extraction unit so that it extracts a volumetrically small portion
of the sample into the diluent. The resulting highly diluted sample may
then be subjected to biological analysis. Those skilled in the art will
recognize that the exact dilution ratio at which there is a transition
from the first embodiment adapted for low range dilution to the second
embodiment adapted for relatively high range dilution may vary depending
on parameters including the exact device geometry, fabrication materials
and sample type.

I. Sensor Cartridge

[0033] A. Low Range Dilution Cartridge Construction

[0034] While the present invention is broadly applicable to assay systems,
it is best understood in the context of the i-STAT® immunoassay
system (Abbott Point of Care Inc., Princeton, N.J., USA), as described in
the jointly-owned pending patent applications and issued patents cited
herein.

[0035] The specific form of the devices, e.g., cartridges, of the present
invention suitable for sample dilution may vary widely. An exemplary
cartridge design according to the first (low range) dilution embodiment
of the present invention is shown in FIGS. 1-6 and comprises a cover 1
(FIGS. 1A and 1B), a base 3 (FIG. 3) and a thin-film adhesive gasket 21
(FIG. 2) disposed between the cover 1 and the base 3. The cartridge also
includes a flexible, e.g., rubberized, pump membrane 9, shown in FIG. 4,
which illustrates an exploded view of the cartridge. FIG. 5 illustrates a
composite drawing of the exemplary cartridge superimposing the features
of the cover, the base and the gasket. FIG. 6 illustrates a conceptual
flow diagram of the fluid and air paths within an immunosensor cartridge
with an integrated sample isolation unit suitable for low range sample
dilution according to one embodiment of the present invention.

[0036] As shown in FIG. 1A, the cover 1 of the cartridge is made of a
rigid material, preferably plastic, capable of repetitive deformation
without cracking at flexible hinge region 10. The cover 1 further
comprises a paddle 7, which is moveable relative to the body of the cover
1, and which is attached to the body by flexible hinge region 10. A pump
opening 6 is disposed in the central region of cover 1, and a recessed
pump membrane region 5 is provided, preferably on the underside of the
cover, as shown in FIG. 1B, for receiving pump membrane 9. Pump membrane
9 may be secured to pump membrane region 5 with an adhesive, which should
form an air-tight seal in order to allow pump membrane 9 to be repeatably
deformed during pumping operations. In other embodiments, not shown, the
membrane may be secured to the outer surface of the cover 1. The
underside of the cover also preferably includes various conduits and
fluid flow features as shown in FIG. 1B and described below.

[0037] The base 3, shown in FIG. 3, includes a closure member 2, attached
to the main body of the base by prongs at the distal end thereof. One
non-limiting embodiment of a closure member is described in jointly-owned
U.S. Pat. No. 7,682,833, the entirety of which is incorporated herein by
reference. The major features of the base include pump cavity 43, diluent
cavity 42, sample entry port 4, and various conduits and fluid flow
features as shown in FIG. 3 and described below. The gasket, shown in
FIG. 2, is disposed between the cover and base and includes various
openings that permit fluids to pass between the conduits in the cover and
conduits in the base.

[0038] In operation, a biological sample, e.g., whole blood, urine, etc.,
is introduced into sample entry port 4 and preferably enters sample
holding chamber 34 (as shown in FIG. 3) passively via capillary action.
The holding chamber 34 extends from the sample entry port 4 to a
capillary stop 25. As shown, capillary stop 25 is formed by a hole within
the gasket (FIG. 2) that separates holding chamber 34 in the base from
analysis conduit 15 in the cover, although in other embodiments, not
shown, the capillary stop may be formed by a constriction in a conduit
either in the base or the cover of the cartridge. A capillary stop is one
example of a sample isolation unit, defined herein as any device capable
of isolating the sample in a specific conduit or region of the device. In
another embodiment, a sample isolation unit may be formed of a sponge or
wicking material that acts to retain the sample.

[0039] After introduction of the sample, the closure member 2 can be
secured, e.g., slidably secured, over the entrance of sample entry port 4
to prevent sample leakage. The cartridge is then inserted into a reading
apparatus in which the sample preferably is automatically manipulated by
actuators to detect the analyte in question. The cartridge is therefore
preferably adapted for insertion into a reading apparatus, and therefore
has a plurality of mechanical and electrical connections adapted for this
purpose. It should also be apparent that partial manual operation of the
cartridge is possible. When operated upon by a pump means within the
reading apparatus, pump membrane 9 exerts a force upon air within an air
bladder comprised of cavity 43, which is covered by pump membrane 9, to
displace fluids within conduits of the cartridge. When operated by a
second pump means, paddle 7 exerts a force upon gasket 21, which can
deform because of slits 22 cut therein. (In an alternative embodiment,
not shown, a second pump membrane may be substituted for paddle 7.)
Gasket 21, in turn, applies pressure on a fluid-containing pouch or
package 57, preferably a foil pack comprising a diluent fluid, that is
disposed within cavity 42. Thus, upon insertion of the cartridge into the
reading apparatus, an actuation mechanism in the reading apparatus
applies pressure to the gasket transmitting pressure onto fluid package
57 filled with, for example, about 20 to 200 μL, e.g., about 160 μL
of diluent in cavity 42, rupturing fluid package 57 upon spike 38, and
expelling diluent into conduit 39, through hole 29 in gasket 21, and into
diluent conduit 53 in the cover. Diluent is transported in diluent
conduit 53 and is optionally metered in a diluent metering chamber, which
is the region within conduit 53 that is between hole 48 (air introduction
port) and hole 47 (diluent introduction port). Preferably, holes 48 and
47 are small enough that the surface tension of the diluent contained
within the diluent conduit 53 inhibits or prevents the diluent from
prematurely passing therethrough. Subsequent pumping action on membrane
9, as described below, allows air to enter the diluent conduit 53 via
hole 48, and expels the diluent, preferably a metered amount of diluent,
through hole 47 and into sample dilution chamber 52. The diluent then
passively mixes with the sample in dilution chamber 52 as the resulting
sample/diluent mixture is expelled through capillary stop 25 with
continued pumping action and into analysis conduit 15.

[0040] In preferred embodiments, the diluent also functions as a wash
fluid and may be separately transported to one or more electrodes in the
cartridge in order to wash unbound species (e.g., unbound analyte and
signal antibodies) from the electrode region after sandwich formation. In
the embodiment shown in FIG. 5, diluent conduit 53 is connected to wash
conduit 20 in order to effect transport of the diluent, acting as wash
fluid, to conduit 20 and ultimately to the electrodes in analysis conduit
15 for washing purposes. As shown, the wash conduit 20 is connected to
the analysis conduit 15 via intervening conduit 8 as shown in FIG. 5. The
length and orientation of conduits 20, 8 and 15 and dilution chamber 52
preferably are designed such that the diluent mixes with the sample and
passes the resulting sample/diluent mixture over the electrodes for
sandwich formation prior to directing of the separate diluent stream,
acting as wash fluid, to the electrodes to wash unbound species
therefrom.

[0041] In some embodiments, not shown, the analyzer mechanism applied to
the cartridge may be used to inject one or more air segments into the
diluent derived from conduit 20 (when the diluent is acting as wash
fluid) at controlled positions within the analysis conduit. These
segments may be used to help wash the sensor surface and the surrounding
conduit using a minimum of fluid. The cover, for example, may further
comprise a hole covered by a thin pliable film for this purpose. In
operation, pressure exerted upon the film expels one or more air segments
into conduit 20 through a small hole 28 in the gasket. See, for example,
U.S. Pat. No. 7,723,099, the entirety of which is incorporated herein by
reference.

[0042] Referring to FIG. 1B, the lower surface of the cartridge cover
further comprises a wash conduit 11, an analysis conduit 15 and a diluent
conduit 53. Optional coatings within one or more of these conduits may
provide hydrophobic surfaces, which may assist in controlling fluid flow
between conduits 11, 20 and 15. A recess 40 in the base provides a
pathway for air to pass from the pump cavity 43 to hole 48 in the gasket,
into conduit 53 in the cover, through hole 47 (diluent introduction port)
in the gasket, and into sample dilution chamber 52 (within sample chamber
34) in the base. In operation, diluent contained in conduit 53 in the
region between hole 48 and hole 47 (diluent introduction port) in the
gasket is pushed through hole 47 and into dilution chamber 52 (a region
within sample chamber 34), which contains a metered sample. In this
manner, the diluent mixes with the metered sample as the two components
are simultaneously pushed through the capillary stop 25 (or other sample
isolation unit) and into analysis conduit 15 for sandwich formation and
analysis.

[0043] As shown in FIG. 2, thin-film gasket 21 comprises various holes and
slits to facilitate transfer of fluid between conduits within the base
and the cover, and to allow the gasket to deform under pressure where
necessary. Hole 122 permits fluid to flow into sample entry port 4 and
into sample holding chamber 34. Hole 24 permits fluid to flow from
conduit 11 into waste chamber 44. Capillary stop 25 comprises an opening
between sample dilution chamber 52 and analysis conduit 15. Hole 28
permits fluid to flow from conduit 19 to waste chamber 44 via optional
closeable valve 41. Holes 30 and 33 permit the plurality of electrodes
that are housed within cutaways 35 and 37, respectively, to contact fluid
within analysis conduit 15. In a specific embodiment, cutaway 37 houses a
ground electrode, and/or a counter-reference electrode, and cutaway 35
houses at least one analyte sensor and, optionally, a conductimetric
sensor. It should be noted that although the conduits described in
connection with the figures variously traverse the gasket, in other
embodiments, the conduits may be oriented substantially in the same plane
without traversing the gasket, or may traverse the gasket in a manner
different than shown in FIGS. 1-5.

[0044] Referring to FIG. 3, sample holding chamber 34 extends from the
sample entry port 4 to capillary stop 25. Sample dilution chamber 52
(FIG. 5) is disposed within sample chamber 34, specifically between hole
47 and capillary stop 25. As shown, the base includes a vent 49 that
facilitates loading of the dilution conduit 53. Specifically, as diluent
is allowed to passively enter diluent conduit 53, air that was contained
in the diluent conduit is allowed to exit therefrom via hole 31 in the
gasket, which is disposed over vent 49. The portion of the sample between
hole 47 (diluent introduction port) and the capillary stop 25 (or other
sample isolation unit) defines a metered volume of sample for dilution.
In exemplary embodiments, the metered sample (prior to dilution) has a
volume of from 0.5 μL to 5 μL, e.g., from 0.1 μL to 10 μL or
from 0.05 μL to 20 μL.

[0045] In accordance with the above description, in one embodiment, the
invention is directed to a sample metering device, comprising a sample
holding chamber oriented between a sample entry port and a sample
isolation unit and having a diluent introduction port disposed
therebetween for introduction of a diluent into the sample holding
chamber. In this embodiment, the volume within the sample holding chamber
between the diluent introduction port and the sample isolation unit
defines a metered volume of a sample for analysis.

[0046] In other embodiments of the invention, not shown, multiple
fluid-containing packages are utilized. In some such embodiments, each
fluid-containing package contains a different fluid, e.g., diluent, a
wash fluid, and/or one or more reagent fluids. An air sac or bladder is
comprised of recess 43, which is sealed on its upper surface by pump
membrane 9. The air bladder is one embodiment of a pump means, and is
actuated by pressure applied to membrane 9, which displaces air in
conduit 40 and thereby displaces the diluent from diluent conduit 53
(optionally metered in a diluent metering chamber) and into sample
dilution chamber 52, where sample dilution occurs, and ultimately
displacing the diluted sample through capillary stop 25 and into analysis
conduit 15. Other types of pumps suitable for use in the present
invention include, but are not limited to a flexible diaphragm, a piston
and cylinder, an electrodynamic pump, and a sonic pump.

[0047] The region between which diluent enters the sample dilution chamber
(e.g., gasket hole 47) from conduit 53 and the capillary stop 25 together
define a predetermined or metered volume of the sample dilution chamber.
An amount of the sample corresponding to this volume together with
diluent from diluent conduit 53 are displaced into the analysis conduit
15 when the air bladder or pump is depressed. This arrangement is,
therefore, one embodiment of a metering means for delivering a metered
amount of an originally unmetered sample into the conduits of the
cartridge.

[0048] Metering may be advantageous, for example, if quantitation of the
analyte is required. In other embodiments, for example when determining
the mere presence of an analyte, metering is not necessary. A waste
chamber 44 is provided for sample and/or fluid that is expelled from the
conduit to prevent contamination of the outside surfaces of the
cartridge. A vent 45 connecting the waste chamber 44 to the external
atmosphere is also provided to facilitate fluid entry into waste chamber
44. A feature of the cartridge of one embodiment of the present invention
is that once a sample is loaded, analysis can be completed and the
cartridge discarded without the operator or others contacting the sample.

[0049] In some embodiments of the invention, a closeable valve is provided
between the analysis conduit and the waste chamber. See, for example, the
materials described in jointly-owned U.S. Pat. No. 7,419,821, which is
referenced above and hereby incorporated by reference in its entirety. In
one embodiment, the valve is comprised of a dried sponge material that is
coated with an impermeable substance. In operation, contacting the sponge
material with the sample or another fluid results in swelling of the
sponge to fill cavity, thereby substantially blocking further flow of
liquid into the waste chamber. The wetted valve also blocks the flow of
air between the analysis conduit and the waste chamber, which permits the
first pump means connected to the sample dilution chamber to displace
fluid within the wash conduit, and to displace fluid from the wash
conduit into the analysis conduit in the following manner. After the
sample is exposed to the sensor for a controlled time, the sample is
moved into the post-analytical conduit where it can be amended with a
reagent. The sample can then be moved back to the sensor and a second
reaction period can be initiated. Alternatively, the post-analysis
conduit can serve simply to separate the sample segment from the sensor.
Within this post-analysis conduit is a single closeable valve, which
connects the air vent of the analysis conduit to the diaphragm air pump.
When this valve closes, the sample is locked in the post analytical
conduit and cannot be moved back to the sensor chip. There are several
different design examples for this valve that are encompassed within the
present invention. Some designs are activated mechanically, while others
activate upon contact with a liquid. Other types of closeable valves that
are encompassed by the present invention include, but are not limited to,
a flexible flap held in an open position by a soluble glue or a gelling
polymer that dissolves or swells upon contact with a fluid or sample thus
causing the flap to close, and alternatively, in one specific embodiment,
a thin layer of a porous paper or similar material interposed between a
conduit and either the waste chamber or ambient air such that the paper
is permeable to air while dry, but impermeable when wet. In the latter
case, it is not necessary that the closeable valve be interposed between
a conduit and the waste chamber, as the valve passes little to no liquid
before closing. Rather, the valve is appropriately placed when positioned
between a conduit and the ambient air surrounding the cartridge. In
practical construction, a piece of filter paper is placed on an opening
in the tape gasket in the fluid path to be controlled. Air can readily
move through this media to allow fluid to be moved through the fluid
path. When the fluid is pushed over this filter, the filter media becomes
filled with liquid and further motion through the fluid path is stopped.
As the filter becomes filled, increasing pressure is required to move
liquid through the pores of the filter. Air flow through the filter is
also minimized or prevented. This valve embodiment requires very little
liquid to actuate the valve, and actuation occurs rapidly and reliably.
Valve materials, dimensions, porosity, wettability, swelling
characteristics and related parameters are selected to provide for rapid
closure, within one second or more slowly, e.g., up to 60 seconds, after
first contact of the valve with the sample. In certain embodiments of the
invention, the closeable valve is a mechanical valve. In one embodiment,
a latex diaphragm is placed in the bottom of the air bladder on top of a
specially-constructed well. The well contains two openings that
fluidically connect the air vent to the sample conduit. As the analyzer
plunger pushes to the bottom of the air bladder, it presses on the latex
diaphragm, which is adhesive-backed, and seals the connection between the
two holes. This blocks the sample air vent and locks the sample in place.

[0050] FIG. 6 is a schematic view of the fluidics within an immunosensor
cartridge in accordance with one embodiment of the present invention.
Regions R1-R8 represent specific immunosensor cartridge components and
C1-C6 represent the fluidic connections between the components. W1
represents a vent, e.g., a wicking vent, which facilitates fluid movement
of diluent from R4 to R3. In particular, R1 is the sample entry port and
associated components for transporting the sample to W1; R2 is the pump
(e.g., air bladder) used to displace the diluent from the diluent conduit
(optionally diluent metering chamber) to a metered volume of sample for
dilution; R3 represents the sample dilution chamber, which terminates in
a sample isolation unit IU (e.g., capillary stop); R4 is the diluent
conduit (optionally comprising a diluent metering chamber); R5 represents
a diluent package and may include a diluent metering chamber; and R6
represents an optional reagent package. R6 represents an optional holding
chamber for a reagent or wash fluid, which may, for example, pass to
conduit 20. The conduit 20, in certain embodiments, can be coated with a
reagent which dissolves into the fluid released from R5 and/or R6. The
reagent amends the fluid so that it can better serve as a wash/analysis
fluid in embodiments where the presence of this reagent in the dilution
fluid in R4 would not be ideal for the assay. In an alternative
embodiment of the invention, C2 can optionally directly link R4 with R6,
such as, for example via a T junction. R7 represents the analysis conduit
and corresponding electrodes; and R8 is a waste chamber.

[0051] As shown in FIG. 7, in some embodiments of the present invention,
the immunosensor cartridge is adapted to a foldable cartridge design of
the type described in jointly-owned U.S. Pat. Appln. No. 61/288,189,
entitled "Foldable Cartridge Housings for Sample Analysis," filed Dec.
18, 2009, the entirety of which are incorporated herein by reference.

[0052] Although the use of capillary stops in a sample metering chamber
are known, the use of a sample isolation unit that comprises a porous
material (e.g., a porous hydrophilic or hydrophobic material, a cellulose
material, nitrocellulose, cotton fiber, paper, glass-filled paper, a
wicking material, a matrix material or other porous material) to form a
metered sample is heretofore unknown.

[0053] Thus, in one embodiment, the invention is to a sample metering
device, comprising a housing comprising a sample chamber located between
a sample entry port and a sample isolation unit, wherein the volume
between the entry port and the sample isolation unit defines a metered
volume of a sample for analysis, and wherein the sample isolation unit
comprises a porous material. Although metered samples thus formed may be
used in the dilution embodiments of the invention, the use of sample
isolation units that comprise a porous material is not limited to the
dilution embodiments discussed herein and may be adopted in immunoassay
devices and methods that do not form diluted samples.

[0054] B. High Range Dilution Cartridge Construction

[0055] In the second dilution embodiment of the invention, a sample is
diluted at a high dilution ratio, e.g., greater than about 50:1. The
specific form of devices, e.g., cartridges, according to this embodiment
of the invention may vary widely. An exemplary cartridge design according
to the second dilution embodiment (high range dilution) of the present
invention is shown in FIGS. 8 and 9. FIG. 8 illustrates a composite
drawing of an exemplary cartridge superimposing the features of the
cover, the base and the gasket according to one embodiment of the
invention, and FIG. 9 provides a flow diagram of the fluid and air paths
within an immunosensor cartridge with an integrated fixed sample
extraction unit in accordance with one embodiment of the present
invention. In various embodiments, dilutions in the range of about 50:1
to about 50,000:1, e.g., from 100:1 to 1,000:1 or from 5,000:1 to
25,000:1, can be performed.

[0056] In many respects, the devices and methods for the high range
dilution embodiments are similar to the low range dilution embodiments.
The primary differences will be highlighted herein, but otherwise the
cartridges preferably are substantially as described above in connection
with the low range dilution embodiments. For example, in the high range
dilution embodiments, a much smaller amount of sample is metered than in
the low range dilution embodiments. In addition, the metering itself is
performed in a different manner. In the high range dilution embodiments,
the metering is conducted at the fixed sample extraction unit, preferably
the distal end thereof, instead of a metered sample that is formed
between two openings (47 & 25 of FIG. 5) that define the sample dilution
chamber (52). Further, in the high range dilution embodiments, diluent
passes over and/or through all or a portion of the fixed sample
extraction unit causing the sample contained therein to be extracted into
the diluent and thereby forming a highly diluted sample.

[0057] As a specific example, high range dilution may be conducted using a
"wick wash" or fixed extraction process design. In this embodiment of the
present invention, sample is introduced into the device and is allowed to
flow, e.g., passively flow through capillary action, until it reaches a
fixed sample extraction unit. After contacting the fluid sample, the
extraction unit preferably becomes saturated with the sample and inhibits
or prevents further flow of the sample. A distal portion of the
extraction unit preferably defines a metered volume of a sample for
dilution. In this embodiment, a very small reproducible amount of sample
(e.g., 100 pL to 2 μL, from 2 nL to 1 μL or from 50 nL to 0.5
μL) is extracted from the fixed sample extraction unit in order to
form a diluted sample.

[0058] As shown in FIG. 8, the immunosensor cartridge includes a sample
entry port 4, a sample introduction chamber 68 and a fixed sample
extraction unit 58. At the distal end of the extraction unit 58 is a
sample dilution chamber 61 where formation of the diluted sample
primarily occurs. As shown, the sample introduction chamber 68 is
oriented in the base and the sample dilution chamber is positioned in the
cover of the cartridge, although in other embodiments both chambers may
be oriented in the cover or in the base. The dilution chamber 61
preferably is oriented proximate, i.e., adjacent, the fixed sample
extraction unit 58 such that as sample is extracted from the fixed sample
extraction unit 58 by diluent, it passes to the sample dilution chamber
for passive mixing 61. As shown, the fixed sample extraction unit 58 is
oriented in the base. In other embodiments, not shown, the fixed sample
extraction unit 58 is oriented in the cover or in both the base and the
cover (extending therebetween). Sample extraction unit 58 is loaded with
sample and defines a metered volume of sample for dilution. A vent 60
(e.g., vent wick) may be provided adjacent the fixed sample extraction
unit 58 to facilitate gas removal as the extraction unit 58 is loaded
with sample.

[0059] An analysis conduit 62 (substantially similar to the analysis
conduit 15 in the low range dilution embodiment) comprising an
analyte-responsive surface and positioned in the cover of the cartridge
is fluidly connected to sample dilution chamber 61. The immunosensor
cartridge further includes a wash conduit 63 for retaining diluent used
to wash or treat the sensor after sandwich formation. The wash conduit 63
is connected to the analysis conduit 62 via intervening conduit 8 in the
base as shown in FIG. 8. A diluent conduit 59 is connected to the wash
conduit 63. Diluent is released from fluid package 57 upon rupturing of
the package on spike 38. The released diluent is then transferred via
conduit 39, through hole 29 in the gasket, and into diluent conduit 59.
In some embodiments, not shown, the fluid package includes multiple
individual fluid packages (e.g., rupturable foil pouches). In this
aspect, each individual fluid package may contain a different fluid
composition, while in other embodiments, one or more of the multiple
pouches may contain the same fluid composition. Each package may have its
own associated pumping mechanism or may share a pumping mechanisms.

[0060] In operation, after the sample has saturated the fixed sample
extraction unit 58, the diluent package 57 is ruptured allowing diluent
to flow into diluent conduit 59 as discussed above. The diluent flows in
diluent conduit 59 until it reaches membrane opening 65 (diluent
introduction port), which optionally comprises a capillary stop. At this
point, pump membrane 9 is actuated causing air to be delivered through
conduit 64 and into diluent conduit 59 via hole 66. The air entering the
diluent conduit causes diluent contained in diluent conduit 59,
specifically between hole 66 and opening 65 (e.g., a metered diluent), to
pass through opening 65 (diluent introduction port) and into contact with
fixed sample extraction unit 58. Upon contact with the extraction unit
58, the diluent acts to extract a small volume of sample therefrom, and
the resulting diluted sample, preferably a highly-diluted sample, is
allowed to pass into sample dilution chamber 61. The diluted sample then
passes to analysis conduit 62. As will be appreciated, the desired
dilution ratio may be obtained by controlling where the dilution conduit
contacts the fixed sample extraction unit and by controlling the volume
of the diluent, e.g., metered diluent in the diluent chamber. Preferably,
the portion that is washed or extracted from fixed sample extraction unit
58 is particularly small, e.g., less than 2 vol. %, less than 0.2 vol. %,
or less than 0.01 vol. %, of the total sample contained in the fixed
sample extraction unit 58 prior to contact with the diluent.

[0061] In addition to diluting the sample, the diluent preferably
functions as a wash fluid as in the low range dilution embodiment
discussed above. In these embodiments, upon rupture of diluent package
57, some diluent is allowed to flow through hole 29 and into wash conduit
63 (wash conduit 63 is shown in fluid communication with diluent conduit
59). Wash conduit 63 is in fluid communication with analysis conduit 62
via intervening conduit 8, in order to allow the diluent, acting as a
wash fluid, to wash any unbound components from the region of the
electrodes.

[0062] In certain embodiments of the present invention, the invention is
to a fixed sample extraction process for high-range dilutions comprising:
loading a fixed sample extraction unit with a sample, wherein the fixed
sample extraction unit is proximate to a sample dilution chamber; washing
(e.g., extracting) a portion of the sample from the extraction unit using
a diluent, preferably a metered volume of diluent, from a diluent conduit
to form a diluted sample; transporting the diluted sample to a sensor;
and performing an analyte assay at the sensor.

[0063] In another embodiment, the fixed sample extraction process further
comprises adding a dilution determinant marker to the sample; measuring
the dilution determinant marker concentration in the sample prior to
introducing said sample into the sample dilution chamber; measuring the
dilution determinant marker concentration in a portion of the sample
washed from the extraction unit; comparing the dilution determinant
marker concentration in the sample prior to introducing said sample into
the sample dilution chamber with the dilution determinant marker
concentration in the portion of the sample washed from the extraction
unit; and calculating the dilution ratio. In still other embodiments, the
fixed sample extraction process includes a step of adding a dilution
determinant marker to the extraction unit; measuring the dilution
determinant marker concentration in a portion of the sample washed from
the extraction unit; and calculating the dilution ratio.

[0064] FIG. 9 is a schematic view of the fluidics within an immunosensor
cartridge with an integrated fixed sample extraction unit in accordance
with one embodiment of the present invention. Regions R1, R2 and R4-R8
represent specific immunosensor cartridge components, C1-C6 represent the
fluidic connections between the components and W1 represents the
controlled dilution device for high-range dilutions (e.g., a fixed sample
extraction unit). In particular, R1 is the sample introduction chamber;
R2 is the pump (e.g., air bladder) used to displace the diluent from the
diluent conduit R4 (e.g., diluent metering chamber) to a metered volume
of sample for dilution; R5 is the diluent package; R6 represents an
optional reagent package; R7 comprises the analysis conduit; and R8 is a
waste chamber. In an alternative embodiment, C2 can optionally directly
link R6 with R4, such as, for example via a T junction.

[0066] In accordance with certain aspects of the present invention, the
materials that form the fixed sample extraction unit or the sample
isolation unit in the low range dilution embodiments preferably are
selected to serve as an effective fluid transport mechanism. Exemplary
materials include any material that may be suitably configured to exhibit
acceptable transport kinetics. To ensure reliable extraction of analyte
from the sample isolation unit or fixed sample extraction unit,
hydrophilic materials or coatings are preferably employed. In some
embodiments, the entire matrix is comprised of a hydrophilic material,
while in other embodiments, only the conduit contact edge and the conduit
walls are so comprised. Exemplary materials for the fixed sample
extraction unit or sample isolation unit include cellulose,
nitrocellulose, cotton fiber, paper and glass-filled paper (e.g.,
Leukosorb®, Pall Corporation, Port Washington, N.Y., USA). In other
embodiments of the invention, this area of the immunosensing device may
be corona treated during assembly to promote hydrophilicity. Use of a
hydrophilic material ensures that bubbles formed in the diluent are not
trapped on the sample isolation unit or fixed sample extraction unit,
thereby impeding analyte transfer. In some embodiments, particularly in
the low range dilution embodiments, as discussed above, the sample
isolation unit may comprise a capillary stop and may not comprise porous
or matrix-type material.

[0067] In embodiments of the invention directed to high-range dilutions,
suitable materials may be less porous (e.g., having a porosity of from 20
μm to 0.1 μm, e.g., from 10 μm to 0.2 μm or from 5 μm to
0.5 μm, as determined by microscopy (e.g., visible or scanning
electron microscopy)), than those materials suitable for relatively
smaller dilutions, the effect of which is that the sample takes longer to
move through the matrix of the fixed sample extraction unit to the
extraction face. In addition, in some embodiments of the invention,
materials generally considered as transverse filter materials (e.g., 0.2
μm water purification filters with a porous outer coating, porous
glasses such as Vycor®, (Corning Incorporated, Corning, N.Y., USA),
treated lateral flow materials from American Filtrona Co. (Richmond, Va.,
USA), filter media from Millipore Corporation (Billerica, Mass., USA),
filter media from Whatman® Schleicher & Schuell® (Maidstone,
Kent, UK, and others) may be used in a lateral mode as the extraction
unit material. In these high dilution embodiments, a small pore volume
relative to a comparatively large volume of diluent is advantageous
(e.g., extraction unit dimensions (length, width, height) of 2 mm×2
mm×100 μm versus 100 μL of diluent) for controlled dilution.
In this aspect, only a sample within a few hundred microns or less is
extracted from the edge of the filter into the diluent as the diluent is
washed over the fixed sample extraction unit. Exemplary volumes of sample
that are incorporated into the diluted sample may range from 50 nL to 0.5
μL, from 2 nL to 1 μL or from 100 pL to 2 μL.

[0068] The porosity of the isolation unit or extraction unit may be
selected to preferentially trap or retard the movement of white and red
blood cells. See, for example, the materials described in jointly-owned
U.S. Pat. No. 5,416,026, which is hereby incorporated by reference in its
entirety. As such, in certain embodiments, the sample that is diluted in
the dilution step may be a plasma fraction rather than whole blood. In
such embodiments, the isolation unit or extraction unit is formatted as a
lateral flow element where blood enters on one side and plasma
predominates towards the other side. This configuration is shown in FIGS.
10A and 10B. In the device of FIG. 10B, a hemolysis detection device
sample orifice 101 for contacting the whole blood sample with dry
separation material 102 is located proximate to the blood entry port 4
(FIG. 8). The plasma or serum fraction wicks along dry separation
material 102, thus becoming separated from whole blood cells. In other
embodiments, the isolation unit or extraction unit also includes a lysing
agent that lyses only the red blood cells in the sample, or both the red
and the white blood cells. Suitable lysing agents may, in some
embodiments, be dry coated onto the isolation unit or extraction unit for
dissolution into the sample. Preferred lysing agents include sodium
deoxycholate and saponin.

[0069] D. Dilution Verification

[0070] In accordance with various embodiments of the present invention,
the effective dilution ratio and reproducibility of any given cartridge
design can be ascertained or confirmed by adding one or more dilution
determinant markers to the sample prior to introduction to the
immunosensing device. In some embodiments, a measurable concentration of
ferricyanide, e.g., from 0.01 to 50, from 0.1 to 10 or from 1 to 5 mM
ferricyanide, is added to the sample. Other electrochemical species
suitable for use as the dilution determinant marker include ruthenium
hexamine and a ferrocene, e.g., ferrocene monocarboxylic acid. In some
exemplary embodiments, the dilution determinant marker is selected from
the group consisting of an electrochemical species, ferricyanide,
ruthenium hexamine, a ferrocene, ferrocene monocarboxylic acid, an
optional dye, fluorescein, an acridinium salt, methylene blue and the
like. Alternative ways of verifying sample dilution include the use of an
optical dye dilution determinant marker (e.g., fluorescein, an acridinium
salt, and methylene blue). In these embodiments, a spectrophotometer may
be used to determine the ratio of concentrations and hence, the dilution
factor. In still other embodiments, dilution can be confirmed using a
sodium ion concentration dilution, e.g., with an integrated sodium ion
sensor with the initial sample sodium concentration verified in a second
cartridge.

[0071] In one embodiment, the invention is to a method of performing an
assay for an analyte in a fluid sample with a cartridge having an
integrated sample dilution element, where the cartridge is adapted for
insertion into a reading apparatus, the method comprising: (a)
introducing a fluid sample into a sample holding chamber of a cartridge
with a sample dilution element, wherein at least a portion of said
dilution element determines a volume of sample for dilution, and wherein
said dilution element further comprises a predetermined known amount of a
dilution determinant marker capable of dissolving into said sample; (b)
pumping a metered volume of diluent from a diluent chamber in said
cartridge, to said sample dilution element to form a diluted sample; (c)
pumping the diluted sample to a sensor in a sensing region of said
cartridge; (d) measuring the concentration of said dilution determinant
marker in said diluted sample; and (e) determining the dilution ratio of
the diluted sample from said measured concentration in step (d) and said
predetermined known amount of step (a). The diluted sample may be at a
dilution ratio of from about 1:1 to 50:1 parts by volume diluent:sample,
or from 50:1 to about 50,000:1 parts by volume diluent:sample. The
dilution ratio value determined in step (e) preferably is used to
calculate the concentration of the analyte in the undiluted sample.

[0072] The predetermined known amount of dilution determinant marker may,
for example, be an embedded value in said reading apparatus, e.g., a
value or coefficient programmed into the instrument software algorithm,
an embedded value on said assay cartridge and automatically read by said
reading apparatus, e.g., a barcode, 2D barcode, magnetic strip, a visible
value, e.g., printed number or letter code, on said assay cartridge and
manually entered into said reading apparatus, or a visible value, e.g.,
printed number or letter code, on said assay cartridge package and
manually entered into said reading apparatus.

[0073] In one embodiment of the invention, a dry reagent, e.g., ferrocene
monocarboxylic acid, is added to the portion of the sample dilution
chamber and parameters are set to give a known dissolved concentration of
ferrocene in the blood sample, e.g., 0.1 to 10 mM or about 1.0 mM. In
certain embodiments, one or more sensors in the cartridge can be used to
detect the ferrocene signal for the diluted sample during the sandwich
formation step. For an intended hundred fold dilution, for example, the
signal may be equivalent to 10 μM ferrocene. During factory
calibration, a current versus concentration algorithm can be established
and embedded in the instrument software. Without being bound by theory,
as ferrocene gives an outer sphere electron transfer reaction, the signal
should not depend on the electrode catalytic activity. Ferrocene
monocarboxylic acid has a half-wave potential about 200 mV more positive
than the p-aminophenol (PAP) used to detect the sandwich, so it should
not provide a material signal, even if the wash step leaves residue.

[0074] In another embodiment, the sample is introduced to the device and
passes onto the sample isolation unit or, in other embodiments of the
invention, to the fixed sample extraction unit. When the dilution
elements are actuated and the diluted sample is passed to the detection
region of the immunosensing device, in certain embodiments of the
invention, a portion of the diluted sample may be manually removed and
tested using a potentiometric sensor or other electrochemical analysis
system for amperometric measurements, a potentiometer for potentiometric
electrochemical tests, or a spectrophotometer to determine the dilution
determinant marker concentration. The ratio of the dilution determinant
marker concentration in the undiluted sample to the dilution determinant
marker concentration in the analyzed portion of the diluted sample
provides the dilution ratio for any given design, and by repeating the
characterization for a set of devices, the precision and accuracy of the
dilution process may be calculated. As such, embodiments of the present
invention provide for independent empirical verification of accuracy and
precision of dilution system designs. Use of the dilution determinant
marker may also serve as a control test during actual use of the
immunosensing device of the present invention.

[0075] While an assay may, in principle, require a defined target dilution
ratio (e.g., 5,000:1), in some embodiments of the present invention, it
may be found that a particular sample isolation unit or fixed sample
extraction unit dilution has high precision but is inaccurate (e.g.,
gives a ratio of 5,300:1). In certain embodiments, the assay coefficients
are adjusted (in this example, to a 5,300:1 ratio) rather than reengineer
the design elements. Those skilled in the art will recognize that for
practical assay development using a single-use cartridge format, this is
one viable approach. Note that while the present disclosure uses integer
ratios for convenience, fractional ratios, e.g., 2:7, 2:7.1, 2:7.01, may
also be within the scope of the invention.

[0076] In preferred embodiments of the invention, the diluent fluid is a
stimulant of plasma without the presence of the analyte. In certain
embodiments, the diluent fluid is aqueous based and includes
electrolytes, buffers and proteins typically found in plasma at high
concentration, e.g., albumin and immunoglobulins. The diluent fluid may
also include lysing agents, stabilizers, and antibacterial agents, which
are well-known in the clinical biochemical arts.

[0077] With regard to the transit time of the diluent fluid in contact
with the controlled dilution device (e.g., time during washing or the
extraction of a portion of the analyte out of the controlled dilution
device), the diluent fluid volume will generally be selected in the range
of about 5 μL to about 200 μL. In some embodiments, the transit
time across the surface (e.g., face or edge) of the controlled dilution
device may be in the range of about 0.1 second to about 100 seconds
(e.g., 1 second to 50 seconds or 2 seconds to 10 seconds).

[0078] In certain embodiments, the diluent fluid is transported through
the conduit and across the surface of the controlled dilution device at a
substantially fixed flow rate, e.g. 10 μL/s. The quicker the diluent
fluid moves, the less time is provided for extraction of analyte from the
dilution device. As such, rate of fluid flow is a control parameter for
the assay system. In some embodiments, the fluid flow can be controlled
by an instrument mechanism and software, which control actuation of the
pump elements. Alternative embodiments to control of flow rate include a
pump cycle that has a fixed stationary dwell time for a portion of the
diluent fluid in contact with the surface of the controlled dilution
device, and also a pump cycle that oscillates a portion of the diluent
across the surface of the controlled dilution device. In these
embodiments, one or more software programs can be utilized to control the
instrument mechanism interaction with the pump elements.

[0079] With regard to the fixed sample extraction unit for high levels of
dilution, in some embodiments, instrument software includes a delay
feature such that the instrument does not deploy the diluent until
sufficient time has elapsed. In a preferred embodiment, the instrument
includes a detector switch that registers the time of insertion to the
test device, and this acts as the t=0 point for the test cycle. Thus, if
the fixed sample extraction unit filling step takes 15 seconds from the
time the sample enters the device, diluent activation is set to be
initiated at t>15 (e.g., t=20 seconds, t=30 seconds or t=60 seconds).

II. Methods of Performing Assays

[0080] The present invention is applicable to methods of performing assays
with a sensor cartridge incorporating an integrated sample dilution
feature and sample metering device. The methods of the invention are
applicable to various biological sample types (e.g., blood, plasma,
serum, urine, interstitial fluid and cerebrospinal fluid).

[0081] In some embodiments, the sensor cartridge is an ion sensor (e.g.,
potentiometric sensor for K, Na, Cl, Ca, NH4 and the like); a
metabolite sensor (e.g., amperometic enzymatic sensor for glucose,
creatinine, cholesterol and the like); an enzyme activity sensor (e.g.,
for liver tests including ALT and AST); and a nucleotide sensor (e.g.,
where amplified target ssDNA forms a sandwich with ssDNA immobilized on
the sensor and other complimentary ssDNA labeled with a signal moiety
such as an enzyme or fluorescent species).

[0082] In preferred embodiments, the present invention may be employed in
one or more of the following areas: immunosensors, most notably in the
context of point-of-care testing; electrochemical immunoassays;
immunosensors in conjunction with immuno-reference sensors; whole blood
immunoassays; single-use cartridge based immunoassays; and non-sequential
immunoassays with only a single wash step; and dry reagent coatings. As
will be appreciated by those skilled in the art, the general concept
disclosed herein is applicable to many immunoassay methods and platforms.
In addition, the present invention is applicable a variety of
immunoassays, including both sandwich and competitive immunoassays.

[0083] After controlled sample dilution and sandwich formation on an
immunosensor, in accordance with various embodiments of the invention,
wash or diluent is deployed. The diluent is preferably advanced through
the connecting conduit and across the sensors by a sequence of small
displacement steps formed by alternating air and fluid segments. In some
embodiments, segment formation is achieved by the instrument applying a
displacement force to actuators in contact with the air bladder and
diluent package in an alternating sequence. This process effectively
entrains a set of air and fluid segments over the sensor. It has been
found that the meniscus between each segment is the most effective part
of the wash cycle to remove sample, unbound analyte and unbound signal
antibody from the sandwich formation or sensor region of the cartridge.
In addition, a sequence of segments provides a more complete wash of the
sensor compared to the same volume of diluent applied in a single pass
over the sensor, although the latter may be used in assays where
non-specific binding is not a significant issue. In preferred
embodiments, the air and fluid segments each have a volume of about 2
μL, but the segment volume can range from less than 1 μL to more
than 20 μL. In certain embodiments of the invention where the analysis
conduit has a cross-sectional area of about 1-2 mm3, each fluid
segment is separated by an air gap of about 2-3 mm. In addition, in some
embodiments, a conductivity sensor may be positioned in the analysis
conduit to monitor the position of fluid-air interfaces and provide
feedback control to the instrument software for pump actuation. (See, for
example, the materials described in jointly-owned U.S. Pat. No.
7,419,821, which is referenced above and hereby incorporated by reference
in its entirety.)

[0084] In alternative embodiments, a segment is injected using a passive
feature. A well in the base of the cartridge is sealed by a tape gasket.
The tape gasket covering the well has two small holes on either end. One
hole is open while the other is covered with a filter material that wets
upon contact with a fluid. The well is filled with a loose hydrophilic
material such as, for example, a cellulose fiber, paper or glass fiber.
The hydrophilic material draws the liquid into the well in the base via
capillary action, displacing the air that was formerly in the well. The
air is expelled through the opening in the tape gasket, creating a
segment whose volume is determined by the volume of the well and the
volume of the loose hydrophilic material. The material used to cover one
of the inlets to the well in the base can be chosen to meter the rate at
which the fluid fills the well and thereby control the rate at which the
segment is injected into the conduit in the cover. This passive feature
permits any number of controlled segments to be injected at specific
locations within a fluid path and requires a minimum of space.

[0085] Within a segment of sample or fluid, an amending substance can be
preferentially dissolved and concentrated within a predetermined region
of the segment. This is achieved through control of the position and
movement of the segment. Thus, for example, if only a portion of a
segment, such as the leading edge, is reciprocated over the amended
substance, then a high local concentration of the substance can be
achieved close to the leading edge. Alternatively, if an homogenous
distribution of the substance is desired, for example if a known
concentration of an amending substance is required for a quantitative
analysis, then further reciprocation of the sample or fluid will result
in mixing and an even distribution.

[0086] In various embodiments of the invention, one or more portions of
the components, conduits, and/or controlled dilution device can be coated
with a dry reagent to amend a sample or fluid. The sample or fluid is
passed at least once over the dry reagent coating to dissolve it.
Reagents used to amend samples or fluid within the cartridge include, but
are not limited to antibody-enzyme conjugates, signal antibodies to the
target analyte, or blocking agents that prevent either specific or
non-specific binding reactions among assay compounds. A surface coating
that is not soluble, but helps prevent non-specific adsorption of assay
components to the inner surfaces of the cartridges can also be utilized
in some embodiments of the present invention.

[0087] As described above, the immunosensor cartridge may further include
an individual diluent package containing a diluent and/or an individual
reagent fluid package containing a reagent fluid. In certain embodiments,
these packages are in the form of a rupturable pouch (e.g., foil pouch).
Manufacture of the rupturable pouches may be performed, for example, as
described in jointly-owned U.S. Pat. Appln. Pub. No. 2010/0068097 A1 to
Ade et al. or in jointly-owned U.S. Pat. No. 5,096,669 to Lauks et al.,
the entirety of each of which is incorporated herein by reference.

[0088] The composition of the diluent is preferably selected to include a
buffer, pH, detergents and the like to promote removal of the unbound
sample and non-specifically bound signal antibody, without substantial
effect on the stability of the sandwich formed on the immunosensor. Those
skilled in the immunoassay art will recognize that such diluent
compositions are well-known, as are methods for their optimization for a
given assay format. In some embodiments of the invention, the volume of
the diluent in the diluent package is selected to be in the range of
about 50 μL to about 200 μL.

[0089] The composition of the detection or reagent fluid is selected to
include an enzyme substrate, buffer, pH, detergents and the like to
promote efficient activity of the enzyme on the signal antibody, without
substantial effect on the stability of the sandwich formed on the
immunosensor. Reagent fluid compositions are well-known in the
immunoassay art, as are methods for their optimization for a given assay
format. In some embodiments, the detection fluid in the detection fluid
package is in the volume range of about 50 μL to about 200 μL and
contains p-aminophenol (PAP) phosphate as a substrate for the alkaline
phosphatase enzyme label in a buffered solution at pH 9.8.

III. Ratiometric Immunoassays

[0090] Given the relative sensitivity of the antibodies that are used and
the actual whole blood concentrations of certain protein molecules (e.g.,
hemoglobin or albumin), it may be desirable to reduce their respective
concentrations, for example, down to the range of about 1 to 100 ng/mL,
which is roughly a 500 to 5000 fold dilution. In these embodiments,
accurate dilution is not critical. Rather, the sample need only be
diluted down to the analyte concentration range where the sensor response
is quasi-linear.

[0091] In certain embodiments, the sample isolation unit approach for
low-range dilutions can be utilized to perform a ratiometric assay in a
blood sample. For example, in one embodiment of the present invention, a
method of performing a ratiometric assay in a blood sample is provided
comprising introducing a blood sample into a sample dilution chamber of a
cartridge, wherein the dilution chamber is located between a diluent
introduction port and a sample isolation unit, and wherein the volume
between the diluent introduction port and the sample isolation unit
defines a metered volume of said sample for dilution. The metered volume
of the sample is diluted with a metered volume of diluent (as described
above) from a diluent conduit located within the cartridge to form a
diluted sample. The diluted sample is pumped to a first and second sensor
in a sensing region (e.g., analysis conduit) of the cartridge, the first
sensor comprising an immunosensor for a first analyte and the second
sensor comprising an immunosensor for a second analyte. A first sandwich
is formed on the first sensor comprising an immobilized first analyte
antibody, the first analyte and a first analyte antibody labeled with a
signaling moiety, and a second sandwich is formed on the second sensor
comprising an immobilized second analyte antibody, the second analyte and
a second analyte antibody labeled with the signaling moiety. The diluted
sample is subsequently washed from the sensing region of the cartridge,
optionally with a wash fluid that is the same composition as the diluent,
and a reagent is introduced for generating a signal from the signaling
moiety to the sensing region of the cartridge. The signal at said first
and second sensors is detected and recorded, and the fractional
percentage of the first analyte to the second analyte is determined from
the signal at said first and second sensors.

[0092] In certain embodiments, the sample isolation unit approach for
high-range dilutions can be utilized to perform a ratiometric assay in a
blood sample. For example, in one embodiment, the invention is to a
method of performing a ratiometric assay in a blood sample, comprising
introducing a blood sample into a sample introduction chamber of a
cartridge, wherein the introduction chamber terminates in a fixed sample
extraction unit, and wherein a distal portion of said extraction unit
defines a metered volume of a sample for dilution. The metered volume of
the sample is diluted with a metered volume of diluent from a diluent
conduit located within the cartridge to form a diluted sample, which is
pumped to first and second sensors in a sensing region of the cartridge.
The first sensor comprises an immunosensor for a first analyte and the
second sensor comprises an immunosensor for a second analyte. A first
sandwich is formed on the first sensor comprising an immobilized first
analyte antibody, the first analyte and a first analyte antibody labeled
with a signaling moiety, and a second sandwich is formed on the second
sensor comprising an immobilized second analyte antibody, the second
analyte and a second analyte antibody labeled with the signaling moiety.
The diluted sample is subsequently washed from the sensing region of the
cartridge, and a reagent for generating a signal from the signaling
moiety is introduced to the sensing region of the cartridge. The signal
at said first and second sensors is detected and recorded and the
fractional percentage of the first analyte to the second analyte is
determined from the signal at said first and second sensors.

[0093] In certain embodiments of the invention, the first analyte
comprises hemoglobin and the second analyte comprises hemoglobin A1c. In
this embodiment, the dilution chamber or the controlled dilution device
preferably comprises a lysing agent (e.g., sodium deoxycholate or
saponin) capable of dissolving in the sample, the diluent or the diluted
sample. In other embodiments where the first analyte comprises albumin
and the second analyte comprises glycosylated albumin, a lysing agent is
not required.

EXAMPLES

[0094] The present invention will be better understood with reference to
the specific embodiments set forth in the following non-limiting
prophetic examples. Suitable non-limiting examples of analytes detectable
with the low dilution format are hemoglobin A1c and C-reactive protein.
Suitable non-limiting examples of analytes detectable with high dilution
format are hemoglobin, human serum albumin and immunoglubulins, e.g., IgG
and IgA. Typical disease states include anemia and immunity assessment.
In addition, detection of beta human chorionic gonadotropin (bHCG) can be
extended into the range of about 50,000 to 500,000 ng/mL.

Example 1

Amperometric Immunoassay

[0095] FIG. 11 illustrates the principle of an amperometric immunoassay
according to specific non-limiting embodiments of the present invention
for determination of C-reactive protein (CRP) 70 in a diluted fluid
sample, a marker of inflammation. A diluted sample (e.g., whole blood
sample) according to the invention, as described above (either high or
low range dilution, but preferably the latter for CRP) is mixed with a
conjugate molecule 71 comprising alkaline phosphatase enzyme (AP)
covalently attached to a polyclonal anti-CRP antibody (aCRP) 72. The
conjugate 71 specifically binds to CRP 70 in the sample, producing a
complex made up of CRP 70 bound to the AP-aCRP conjugate 71. In a capture
step, this complex binds to the capture aCRP antibody 72 attached onto
the surface of or unattached but proximate to the sensor (e.g., gold
electrode 74). In some embodiments, a conductivity sensor (not shown) is
used to monitor when a certain volume of the sample (e.g., sample
segment) reaches the sensor. The time of arrival of the sample can be
used to detect leaks within the cartridge (e.g., a delay in arrival
signals a leak). The position of the sample segment within the analysis
conduit can be actively controlled using the edge of the fluid sample as
a marker. As the sample/air interface crosses the conductivity sensor, a
precise signal is generated that can be used as a fluid marker from which
controlled fluid excursions can be executed. The sample segment is
preferentially oscillated edge-to-edge over the sensor 74 in order to
present the entire sample to the sensor surface. A second reagent can be
introduced in the analysis conduit beyond the sensor, which becomes
homogenously distributed during the fluid oscillations. The sensor chip
contains a capture region or regions coated with antibodies for the
analyte of interest. These capture regions are defined by a hydrophobic
ring of polyimide or another photolithographically produced layer. A
microdroplet or several microdroplets (5-40 nL in size) containing
antibodies in some form, e.g., bound to latex microspheres, is dispensed
on the surface of the sensor. The photodefined ring contains the one or
more aqueous droplets, allowing the antibody coated region to be
localized to a precision of a few microns. In some embodiments, the
capture region can be made from 0.03 mm2 to 2 mm2 in size. The
upper end of this size range is limited by the size of the conduit and
sensor in certain embodiments, and is not a limitation of the invention.

[0096] Thus, the gold electrode 74 is coated with a biolayer 73 comprising
a covalently attached anti-CRP antibody, to which the CRP/AP-aCRP complex
binds. AP is thereby immobilized close to the electrode in proportion to
the amount of CRP initially present in the sample. In addition to
specific binding, the enzyme-antibody conjugate may bind non-specifically
to the sensor. Non-specific binding provides a background signal from the
sensor that is undesirable and preferably is minimized. As described
above, the rinsing protocols, and in particular the use of segmented
fluid to rinse the sensor, provide efficient means to minimize this
background signal. In a second step subsequent to the rinsing step, a
substrate 75 that is hydrolyzed by, for example, alkaline phosphatase to
produce an electroactive product 76 is presented to the sensor. The
amperometric electrode is either clamped at a fixed electrochemical
potential sufficient to oxidize or reduce a product of the hydrolyzed
substrate but not the substrate directly, or the potential is swept one
or more times through an appropriate range. Optionally, a second
electrode may be coated with a layer where the complex of CRP/AP-CRP is
made during manufacture, to act as a reference sensor or calibration
means for the measurement.

[0097] In the present example, the sensor comprises two amperometric
electrodes that are used to detect the enzymatically produced
4-aminophenol from the reaction of 4-aminophenylphosphate with the enzyme
label alkaline phosphatase. The electrodes are preferably produced from
gold surfaces coated with a photodefined layer of polyimide. Regularly
spaced openings in the insulating polyimide layer define a grid of small
gold electrodes at which the 4-aminophenol is oxidized in a 2 electron
per molecule reaction.

H2N--C6H4--OH→HN═C6H4═O+2H.sup.+-
+2e.sup.

[0098] Sensor electrodes further comprise a biolayer, while reference
electrodes can be constructed, for example, from gold electrodes lacking
a biolayer, or from silver electrodes, or other suitable material.
Different biolayers can provide each electrode with the ability to sense
a different analyte.

[0099] Substrates, such as p-aminophenol (PAP) species, can be chosen such
that the E1/2of the substrate and product differ substantially.
Preferably, the E1/2 of the substrate is substantially higher than
that of the product. When the condition is met, the product can be
selectively electrochemically measured in the presence of the substrate.
In specific embodiments, the substrate is comprised of a phosphorylated
ferrocene or, more preferably, phosphorylated PAP.

[0100] The size and spacing of the electrode play an important role in
determining the sensitivity and background signal. The important
parameters in the grid are the percentage of exposed metal and the
spacing between the active electrodes. The position of the electrode can
be directly underneath the antibody capture region or offset from the
capture region by a controlled distance. The actual amperometric signal
of the electrodes depends on the positioning of the sensors relative to
the antibody capture site and the motion of the fluid during the
analysis. A current at the electrode is recorded that depends upon the
amount of electroactive product in the vicinity of the sensor.

[0101] The detection of alkaline phosphatase activity in this example
relies on a measurement of the 4-aminophenol oxidation current. This is
achieved at a potential of about +60 mV versus the Ag/AgCl ground chip.
The exact form of detection used depends on the sensor configuration. In
one version of the sensor, the array of gold microelectrodes is located
directly beneath the antibody capture region. When the diluent is pulled
over this sensor, enzyme located on the capture site converts the
4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.
The concentration of the 4-aminophenylphosphate is selected to be in
excess, e.g., 10 times the Km value. The analysis solution is 0.1 M in
diethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. Additionally, the
analysis solution contains 0.5 mM MgCl, which is a cofactor for the
enzyme.

[0102] In another electrode geometry embodiment, the electrode is located
a few hundred microns away from the capture region. When a fresh segment
of diluent is pulled over the capture region, the enzyme product builds
with no loss due to electrode reactions. After a time, the solution is
slowly pulled from the capture region over the detector electrode,
resulting in a current spike from which the enzyme activity can be
determined.

[0103] An important consideration in the sensitive detection of alkaline
phosphatase activity is the non-4-aminophenol current associated with
background oxidations and reductions occurring at the gold sensor. Gold
sensors tend to give significant oxidation currents in basic buffers at
these potentials. The background current is largely dependent on the
buffer concentration, the area of the gold electrode (exposed area),
surface pretreatments and the nature of the buffer used. Diethanolamine
is a particularly good activating buffer for alkaline phosphatase. At
molar concentrations, the enzymatic rate is increased by about three
times over a non-activating buffer such as carbonate.

[0104] In alternative embodiments, the enzyme conjugated to an antibody or
other analyte-binding molecule is urease, and the substrate is urea.
Ammonium ions produced by the hydrolysis of urea are detected in this
embodiment by the use of an ammonium sensitive electrode.
Ammonium-specific electrodes are well-known to those of skill in the art.
A suitable microfabricated ammonium ion-selective electrode is disclosed
in U.S. Pat. No. 5,200,051, which is referenced above and hereby
incorporated by reference in its entirety. Other enzymes that react with
a substrate to produce an ion are known in the art, as are other ion
sensors for use therewith. For example, phosphate produced from an
alkaline phosphatase substrate can be detected at a phosphate
ion-selective electrode.

[0105] Referring now to FIG. 12, there is illustrated the construction of
an embodiment of a microfabricated immunosensor. Preferably, a planar
non-conducting substrate is provided 80, onto which is deposited a
conducting layer 81 by conventional means or microfabrication, known to
those of skill in the art. The conducting material is preferably a noble
metal such as gold or platinum, although other unreactive metals such as
iridium may also be used, as may non-metallic electrodes of graphite,
conductive polymer, or other materials. An electrical connection 82 is
also provided. A biolayer 83 is deposited onto at least a portion of the
electrode. In the present disclosure, a biolayer refers to a porous layer
comprising on its surface a sufficient amount of a molecule 84 that can
either bind to an analyte of interest, or respond to the presence of such
analyte by producing a change that is capable of measurement. Optionally,
a permselective screening layer may be interposed between the electrode
and the biolayer to screen electrochemical interferents as described in
U.S. Pat. No. 5,200,051, which is referenced above and hereby
incorporated by reference in its entirety.

[0106] In some embodiments of the present invention, a biolayer is
constructed from latex beads of specific diameter in the range of 0.001
μm to 50 μm. The beads are modified by covalent attachment of any
suitable molecule consistent with the above definition of a biolayer.
Many methods of attachment exist in the art, including providing amine
reactive N-hydroxysuccinimide ester groups for the facile coupling of
lysine or N-terminal amine groups of proteins. In specific embodiments,
the biomolecule is chosen from among ionophores, cofactors, polypeptides,
proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,
lectins, neurochemical receptors, oligonucleotides, polynucleotides, DNA,
RNA, or suitable mixtures. In more specific embodiments, the biomolecule
is an antibody selected to bind one or more of human chorionic
gonadotrophin, C-reactive protein, hemoglobin, hemoglobin A1c, IgG, IgA,
brain natriuretic peptide (BNP), troponin I, troponin T, troponin C, a
troponin complex, creatine kinase, creatine kinase subunit M, creatine
kinase subunit B, myoglobin, myosin light chain, or modified fragments of
these. Such modified fragments are generated by oxidation, reduction,
deletion, addition or modification of at least one amino acid, including
chemical modification with a natural moiety or with a synthetic moiety.
Preferably, the biomolecule binds to the analyte specifically and has an
affinity constant for binding analyte ligand of about 107 to
1015 M-1.

[0107] In one embodiment, the biolayer, comprising beads having surfaces
that are covalently modified by a suitable molecule, is affixed to the
sensor by the following method. A microdispensing needle is used to
deposit onto the sensor surface a small droplet, preferably about 0.4 nL,
of a suspension of modified beads. The droplet is permitted to dry, which
results in a coating of the beads on the surface that resists
displacement during use.

[0108] In addition to immunosensors in which the biolayer is in a fixed
position relative to an amperometric sensor, the present invention also
envisages embodiments in which the biolayer is coated upon particles that
are mobile. In certain embodiments, the cartridge can contain mobile
microparticles capable of interacting with an analyte, for example
magnetic particles that are localized to an amperometric electrode
subsequent to a capture step, whereby magnetic forces are used to
concentrate the particles at the electrode for measurement. See, for
example, jointly-owned U.S. patent application Ser. No. 12/815,132 and
U.S. Provisional Pat. Appln. Ser. Nos. 61/371,066; 61/371,109;
61/371,077; and 61/371,085. Each of these patent applications is hereby
incorporated by reference in its entirety. One advantage of mobile
microparticles in the present invention is that their motion in the
sample or fluid accelerates binding reactions, making the capture step of
the assay faster. For certain embodiments using non-magnetic mobile
microparticles, a porous filter is used to trap the beads at the
electrode.

[0109] Referring now to FIG. 13, there is illustrated a mask design for
several electrodes upon a single substrate in accordance with one
embodiment of the present invention. By masking and etching techniques,
independent electrodes and leads can be deposited. Thus, a plurality of
immunosensors, 94 and 96, and conductimetric sensors, 90 and 92, are
provided in a compact area at low cost, together with their respective
connecting pads, 91, 93, 95, and 97, for effecting electrical connection
to the reading apparatus. In principle, a very large array of sensors can
be assembled in this way, each sensitive to a different analyte or acting
as a control sensor.

[0110] In specific embodiments of the present invention, immunosensors are
prepared as follows. Silicon wafers are thermally oxidized to form an
insulating oxide layer having a thickness of about 1 μm. A
titanium/tungsten layer is sputtered onto the oxide layer to a preferable
thickness of between 100-1000 Å, followed by a layer of gold that is
most preferably 800 Å thick. Next, a photoresist is spun onto the
wafer and is dried and baked appropriately. The surface is then exposed
using a contact mask, such as a mask corresponding to that illustrated in
FIG. 13. The latent image is developed, and the wafer is exposed to a
gold-etchant. The patterned gold layer is coated with a photodefinable
polyimide, suitably baked, exposed using a contact mask, developed,
cleaned in an O2 plasma, and preferably imidized at 350° C.
for about 5 hours. The surface is then printed with antibody-coated
particles. Droplets, preferably of about 0.4 nL volume and containing 2%
solid content in deionized water, are deposited onto the sensor region
and are dried in place by air drying. Optionally, an antibody
stabilization reagent (e.g., StabilCoat® SurModics, Inc., Eden
Prairie, Minn., USA) is overcoated onto the sensor. Drying the particles
causes them to adhere to the surface in a manner that prevents
dissolution in either sample or fluid containing a substrate. This method
provides a reliable and reproducible immobilization process suitable for
manufacturing sensor chips in high volume.

Example 2

Immunosensing Device and Method of Use

[0111] The present example describes one of the methods of use of a
cartridge embodied in the present invention. In this embodiment, the
cartridge includes a closeable valve, located between the immunosensor
and the waste chamber. For a CRP assay, a blood sample is first
introduced into the sample chamber of the cartridge. In the following
time sequence, time zero (t=0) represents the time at which the cartridge
is inserted into the cartridge reading device. Times are given in
minutes. Between t=0 and t=1.5, the cartridge reading device makes
electrical contact with the sensors through electrical contact pads and
performs certain diagnostic tests. Insertion of the cartridge perforates
the foil pouch introducing diluent into the wash conduit, as previously
described, as well as into the diluent conduit. The diagnostic tests
determine whether fluid or sample is present in the conduits using the
conductivity electrodes, determine whether electrical short circuits are
present in the electrodes, and ensure that the sensor and ground (e.g.,
reference/counter) electrodes are thermally equilibrated to, preferably,
37° C. prior to the analyte determination.

[0112] Between t=0.5 and t=1.5, the pumping means pumps a metered diluent
from the diluent conduit into a dilution chamber, where the diluent is
mixed with a metered portion of the sample to form a diluted sample.

[0113] Between t=1.5 and t=6.75, the diluted sample, preferably between
about 4 μL and about 200 μL, more preferably between about 4 μL
and about 20 μL, and most preferably about 7 μL, is used to contact
the sensor. The edges defining the forward and trailing edges of the
diluted sample are reciprocally moved over the conductivity sensor region
at a frequency that is preferably between 0.2 to 5.0 Hz, and is most
preferably 0.7 Hz. During this time, the enzyme-antibody conjugate and
beads (e.g., mobile beads or magnetically-susceptible beads) dissolve
within the sample. The amount of enzyme-antibody conjugate that is coated
onto the conduit is selected to yield a concentration when dissolved that
is preferably higher than the highest anticipated CRP concentration, and
is most preferably six times higher than the highest anticipated CRP
concentration in the sample.

[0114] Between t=6.75 and t=10.0, the diluted sample is moved to the
immunosensor for capture of the beads. As shown in FIGS. 1-4, the sample
is moved into the waste chamber via closeable valve 41, wetting the
closeable valve and causing it to close. The seal created by the closing
of the valve 41 permits the first pump means to be used to control motion
of fluid from conduit 11 to analysis conduit 15. After the valve 41
closes and the remaining sample is locked in the post analysis conduit,
the analyzer plunger retracts from the flexible diaphragm of the pump
means, creating a partial vacuum in the analysis conduit. This forces the
diluent through the small hole in the tape gasket 21 and into a short
transecting conduit 8 in the base, and then up through another hole in
gasket 21 and into analysis conduit 15 (in cover 1). The diluent is then
pulled further and the front edge of the diluent (acting here as wash
fluid) is oscillated across the surface of the immunosensor chip in order
to shear the sample near the walls of the conduit. The conductivity
sensor on the chip is used to control this process.

[0115] The efficiency of the wash is optimally further enhanced by
introduction into the fluid of one or more menisci or air segments. The
air segments may be introduced by either active or passive means. Fluid
is then forcibly moved towards the sensor chip by the partial vacuum
generated by reducing the mechanical pressure exerted upon pump membrane
9, causing the analysis conduit 15 in the vicinity of transecting conduit
8 to fill with diluent as wash fluid. This region of the analysis conduit
optionally has a higher channel height resulting in a meniscus with a
smaller radius of curvature. The region of the analysis conduit in the
direction of the one or more sensors optionally has a conduit height is
that is smaller. In one aspect, the diluent passively flows from the
region adjacent the transecting conduit 8 towards this low height region
of the analysis conduit, thereby washing the conduit walls. This passive
wicking effect allows further effective washing of the analysis conduit
using a minimal volume of fluid and without displacing the beads that are
attached to the sensor. In this embodiment, the fluid located within the
wash conduit may also contain a substrate for the enzyme. In other
embodiments, amendment of the fluid using dried substrate within the wash
conduit may be utilized.

[0116] Following the positioning of a final segment of fluid over the
sensor, measurement of the sensor response is recorded and the
concentration of analyte is determined. Specifically, at least one sensor
reading of a sample is made by rapidly placing over the sensor a fresh
portion of fluid containing a substrate for the enzyme. Rapid
displacement both rinses away product previously formed, and provides a
new substrate to the electrode. Repetitive signals are averaged to
produce a measurement of higher precision, and also to obtain a better
statistical average of the baseline, represented by the current
immediately following replacement of the solution over the immunosensor.

[0117] The invention described and disclosed herein has numerous benefits
and advantages compared to previous devices. These benefits and
advantages include, but are not limited to ease of use, the automation of
most if not all steps of the analysis, which eliminates user included
error in the analysis. While the invention has been described in terms of
various preferred embodiments, those skilled in the art will recognize
that various modifications, substitutions, omissions and changes can be
made without departing from the spirit of the present invention.
Accordingly, it is intended that the scope of the present invention be
limited solely by the scope of the following claims.